The terminal inverted repeats of IS911: Requirements for synaptic complex assembly and activity

The bacterial insertion sequence IS911 transposes via a covalently closed c ircular intermediate. Circle formation involves transposase-mediated pairin g of both insertion sequence ends. While full-length transposase, OrfAB, bi nds poorly in vitro to IS911 DNA fragments carrying a copy of the IS911 end , truncated protein derivatives carrying the first 135 (OrfAB[1-135]) or 14 9 (OrfAB[1-149]) amino acid residues bind efficiently. They generate a pair ed-end complex containing two such fragments which resembles that expected for the first synaptic complex. Shorter protein derivatives lacking a regio n involved in multimerisation do not form these complexes but modify the bi nding of OrfAB[1-135] and OrfAB[1-149]. DNaseI footprinting demonstrated th at OrfAB[1-149] protects a sub-terminal (internal) region of the inverted r epeats which includes two blocks of sequence (beta and gamma) conserved bet ween the left (IRL) and right (IRR) ends. DNA binding assays in vitro and m easurement of recombination activity in vivo of sequential deletion derivat ives of the two inverted repeats suggested a model in which the N-terminal region of OrfAB binds the conserved boxes beta and gamma in a sequence-spec ific manner and anchors the two insertion sequence ends into a paired-end c omplex. The external region of the inverted repeat is proposed to con tact the C-terminal transposase domain carrying the catalytic site. (C) 2001 Aca demic Press.